Microscope slide cover with integrated reservoir

ABSTRACT

A cover ( 10 ) for substrate (not shown) including: a body ( 12 ) defining a cavity ( 18 ), for positioning over the substrate (not shown) to form a reaction chamber ( 18 ); and a projection ( 13 ) extending from the body ( 12 ) to define a fluid reservoir ( 14 ), when the cover ( 10 ) is fitted to the substrate (not shown), the fluid reservoir ( 14 ) being in fluid communication with the cavity ( 18 ).

FIELD OF THE INVENTION

The present invention relates to a cover for a substrate, and in oneform a cover for use with a microscope slide.

BACKGROUND OF THE INVENTION

Microscope slides are commonly used to view samples of material under amicroscope. The samples may contain human tissue, and may requiretreatment such as staining, so that properties of the sample can beidentified. Other materials such as DNA, RNA, or proteins may beincluded on the slide.

It is common for several reactions to be undertaken on a sample on aslide. Once the reactions have taken place the slide may be viewed undera microscope. Performing the reactions on the slide can be difficult toautomate, as the tissue samples require careful preparation and certainreactions require carefully controlled environments.

SUMMARY OF THE PRESENT INVENTION

In accordance with the present invention, there is provided a cover fora substrate including:

-   a body defining a cavity, for positioning over the substrate to form    a reaction chamber; and-   a projection extending from the body to define a fluid reservoir,    when the cover is fitted to the substrate, the fluid reservoir being    in fluid communication with the cavity.

Preferably the cavity extends the full width of a sample holding regionon the substrate.

Preferably, a protrusion extends from the projection, to assist inwicking fluid into the reservoir.

Preferably, the reservoir is defined between the projection, which isspaced from the substrate, and legs located at sides edge of the cover.

In one form the projection is formed front two sections, the firstsection is angled at least at substantially 60° relative to the cavityand the second section is angled at least at substantially 15°.

In one form, the cover further includes a second reservoir, at anopposite end of the cover.

Preferably wall portions are located at the edge of the cover,surrounding the cavity on two or more sides.

In one form the legs extend along the sides of the cover to form thewall portions.

In a preferred form, the cover includes a locator for controlling andlocating the cover, the locator being arranged at an end of the coveropposite the projection.

In one form the cavity extends to an end edge of the cover adjacent thelocator.

In one form the cover is supported on the substrate by the wallportions.

Preferably, the cover is made from a polymer material.

In one form the cavity includes a coating of reduced surface roughnessthan the polymer material.

In another form the cavity includes a coating with reduced porosity.

In another form the cavity has one or more coatings.

Preferably a first coating is a material having similar properties tothe material of the slide.

Preferably the first coating is silicon dioxide.

Preferably a second coating is placed intermediate a first coating toprovide improved contact properties between the cover and first coating.

Preferably, the cover has associated wing structures that allow thecover to be engaged and pivoted relative to the substrate so as to openthe reaction chamber and allow the slide to be cleared of fluid.

In another aspect, there is provided a combination of a substrate and acover, as described above, wherein the cavity of the cover is arrangedto face the substrate so as to form a reaction chamber.

In yet another aspect, there is provided a method of treatment of asample on a sample holding region of a substrate, including locating acover, as described above, over the substrate, so that the cavity of thecover faces the substrate to form a reaction chamber over the sampleholding region, and depositing fluid into the fluid reservoir to allowthe fluid to be drawn into the reaction chamber, as required.

Preferably, the method further includes sliding the cover relative tothe substrate to vary a degree of overlap between the cover and thesample holding region, which results in a corresponding variation in thereaction chamber volume.

Preferably, the method further includes sliding the cover relative tothe substrate until wing structures associated with the cover areengaged and lifted relative to the substrate to pivot the cover into anopen condition, and allow fluid to drain from the reaction chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described, by way of non-limiting example only, withreference to the accompanying drawings, in which:

FIG. 1 shows an example of a microscope slide;

FIGS. 2 (a)-(c) show top, side and bottom views of a first example of acover for a slide;

FIG. 3 shows a perspective view of the cover of FIG. 2;

FIGS. 4 (a)-(c) show farther views of the cover of FIG. 2 located on theslide of FIG. 1;

FIG. 5 is a perspective view of the cover and slide arrangement of FIG.4, showing a cutaway section of the cover;

FIG. 6 shows a schematic cross section of the cover and slide of FIG. 5;

FIG. 7 shows a perspective view of a tray adapted to locate covers andslides;

FIGS. 8 (a) and (b) show schematic top and sectional side views,respectively, of a further example of a cover;

FIGS. 9 (a) and (b) show schematic top and sectional side views,respectively, of a further example of a cover;

FIGS. 10 (a) and (b) show schematic top and sectional side views,respectively, of a further example of a cover;

FIGS. 11 (a) and (b) show schematic top and sectional side views,respectively, of a further example of a cover,

FIGS. 12 (a) and (b) show schematic top and sectional side views,respectively, of a further example of a cover;

FIGS. 13 (a) and (b) shows schematic top and sectional side views,respectively, of a further example of a cover;

FIGS. 14 (a) and (b) show top and bottom perspective views,respectively, of a further example of a cover;

FIG. 15 shows a schematic side view of a nose portion of a cover;

FIG. 16 (a) and (b) show schematic top and sectional side views,respectively, of a further example of a cover;

FIG. 17 shows a schematic side view of a further example of a noseportion of a cover;

FIG. 18 shows the cover of FIG. 2 mounted to the tray of FIG. 7;

FIGS. 19 (a)-(c) shows the cover of FIG. 2 in various positions over theslide of FIG. 1; and

FIGS. 20 (a) and (b) show a bottom perspective view and enlarged partialperspective view, respectively of a modified cover.

DETAILED DESCRIPTION

A microscope slide 1 is shown in FIG. 1 as including an upper surface 2containing a sample 3. The slide 1 is identified by a unique bar code 4.The sample 3, such as a thinly sliced tissue section, is located on theslide 1 in a sample holding region 5, which needs to be covered by acover, such as shown in FIG. 2, for subsequent application of testfluids and the like.

FIGS. 2 (a)-(c) and FIG. 3 show a cover 10 as having a body 12, a fluidreceiving zone 14, a locating means 16 and a cavity 18 on an undersideface 19. Surrounding the cavity 18 on two sides is a wall portion 20. Atone end of the cover 10, the wall portion 20 joins with legs 21 whichextend upwardly and away from the face 19. The legs 21 are spanned by aprojection 13 which defines a fluid reservoir 17, between an undersideof the projection and the legs 21.

The cover 10 is shown fitted to a slide 1 in FIGS. 4 and 5. The fluidreservoir 17 is shown roost clearly in FIG. 4 (c) where a detailed viewof part of a section A-A taken across the cover 10 and slide 1 isillustrated. The projection 13, with leg 21 at either end, is raisedrelative to the slide 1, to form a volume capable of holding fluiddispensed onto the slide 1. In this way fluid reservoir 17 enables fluiddispensed onto slide 1 to be held until required, without spilling offan edge of the slide. The projection 13 further assists in spreading thefluid across the full width of the cavity

The overlap of the cavity 18 with the slide 1 forms what may bedescribed as a reaction chamber, as illustrated in FIG. 6. The cavitymay vary according to application, typically from 20-200 microns. Thewall portion 20 is adapted to support the cover on the slide 1. Thecavityed face 22, wall portion 20 and sample holding region 5 of a slide1 form a reaction chamber 24 when the cover 10 is placed at leastpartially over the sample holding region 5.

The fluid reservoir 17 is typically sized to be larger than the volumeof the reaction chamber 24, for example 150% of the volume of thereaction chamber. This provides sufficient volume of fluid to fill thereaction chamber completely, while allowing some excess to flush thechamber, and an amount to be retained in the fluid reservoir to providea reservoir for evaporation.

Clamping forces may also be applied to the cover once loaded onto theslide, and these forces are designed to provide a seal between the wallportions 20 and the upper surface of the slide 1. This is to restrictfluid leakage from the side of the cover. In one example (not shown) thewall portions may have an additional member to assist sealing of thewall portions with the upper surface 2 of the slide 1. This additionalmember may be a softer polymer or rubber material.

The cover 10 also includes engaging surfaces in the form of wings 26.The wings 26 are adapted to engage ramps 28 of a tray 21 shown in FIG.7, to thereby lift the cover clear of the surface of the slide 1. Anexample of the wings lifting the cover free is shown more clearly inFIG. 18. The cover 10 may be controlled by an arm (not shown) moving thelocating means 16. The cover 10 may be placed in a number of positionsover the slide, exemplified by the positions of the cover relative tothe slide shown in FIG. 19. In FIG. 19 (a), the cover 10 is in an openposition relative to the slide 1, as the sample is exposed and open.FIG. 19 (b) shows the cover in a partially closed position, and FIG. 19(c) shows the cover in a fully closed position, where the sample iscompletely covered by the cover and is therefore wholly contained withinthe reaction chamber 24. The reaction chamber formed by the cover andcavity 18, as shown in FIG. 5, extends over most of the slide 1. Howeverit is possible that the sample may be placed more towards the end of theslide distal from the bar code 4, and therefore a smaller reactionchamber 24 is required. Reducing the size of the reaction chamber 24reduces the amount of fluid required to fill the chamber, which can beimportant where expensive or scarce fluids are used. It is possible toform a smaller reaction chamber with the cover 10, by only covering aporion of the slide 1 with the cover 10. This position is shown in FIG.18 (b).

Variations in cover constructions are schematically shown in FIGS. 8-17.In FIGS. 8-17, only the front segments of the covers are shown, and thelocating means 16 have been omitted from view for clarity and like partsare denoted by like reference numerals.

In FIG. 8 (a) a cover 10 is shown having a body 12, projecting legs 21,a protruding section 13 and an indent 30. The projecting legs 21 eitherside of the body 12 form a fluid receiving zone 14. When placed onto aslide, fluid may be dispensed into the fluid receiving zone, where itspreads in a circular fashion to contact the protruding section 13. Theindent 30 allows the fluid to contact a wider portion of the protrudingsection 13 than if the front edge of the protruding section was straight(as shown in FIG. 9). Once the fluid is in contact with the protrudingsection 13, it wicks across the width of the cavity 18. If suction isapplied at the rear of the cavity, or the cover is moved along the slidefrom an open position to a more closed position, then the fluid beginsto fill the cavity 18. When the cavity 18 has moved across the sample 3,it forms the reaction chamber 24 as the fluid may react with the sample3.

FIGS. 9 (a) and (b) show a more simple construction of a cover 10 thatmay be used in some circumstances. The operation of the cover 10 is thesame as the operation of the cover 10 in FIGS. 8 (a) and (b).

FIG. 10 s (a) and (b) show a cover 10 having a body 12 with projectinglegs 21. A protruding section 13 and a bar 31 surround a fluid receivingzone 14 for receiving fluid. The fluid may be dispensed onto theprotruding section 13, where it flows down and onto the slide surface 2.The protrusion 13 and bar 31 cause the fluid to spread across the widthof the cavity 18, enabling the cavity to be filled with fluid.

The covers 10 of FIGS. 11, 12 and 16 operate in similar ways to thosedescribed above.

In relation to all of the above-described coves, it should beappreciated that the covers are generally 25 mm across, and the cavity18 is typically only 20-200 micrometres high. As such, overall fluiddispense volumes may be in the order of 20-300 microlitres.

FIG. 13 (a) shows another cover 10 having a body 12, legs 21 and a fluiddispenser 100 dispensing fluid 102 onto the slide 1. In FIG. 13 (a), thefluid 102 has already been dispensed, and has formed a fluid reservoirin the fluid reservoir 17. The schematic Figure shows a typical wickingpattern formed by the fluid as it contacts the cover 1. In FIG. 13 (b),the fluid is just being dispensed onto the projection 13. In the volumesdispensed, the fluid forms a pool of comparable size to some of thecover features. Not only does the fluid flow forward of the cover asshown in FIG. 13 (a), but it also flows under the cover to at leastpartially fill cavity 18. As mentioned above the fluid may be drawn intothe cavity further by movement of the cover over the slide or suctionapplied to the rear of the cavity 18.

FIG. 14 (a) and (b) shows a further embodiment of a cover 10 where likereference numerals are again used to denote like ports. The cover has afluid reservoir 17, a projection 13, and a protrusion in the form of nib15. Fluid may be deposited directly On the nib 15 so that the fluidrolls over the projection 13 into reservoir 14, and to the cavity 18, asrequired. If fluid is placed too far ahead of the cover, there arecircumstances that may cause the fluid to reach the edge of the slidebefore wicking across the width of the cavity 18. It has been found thatusing the projection 13 causes the dispensed fluid to contact thecovertile and spread along the full width of the cavity 13, due to thepositive attraction of the covertile and the fluid. The capillary forcesin the cavity cause the fluid to spread out, and the reservoir holdssufficient fluid to ensure that fluid dispensed onto the slide at leastfills the cavity 18. The nib 15 is useful in that if the pipette is notplaced to dispense the fluid accurately onto the slide, and for examplemisses a few millimetres in front of the projection, the nib 15 will belikely to contact the fluid, which will be drawn to the protrusion andinto the reservoir. This assists in reducing bubble or void formationwithin the cavity. The nib 15 may extend approximately 1-5 mm from theprojection 13.

FIG. 17 shows an example of how fluid spreads across a slide whendeposited in front of a cover 50. A variety of profiles for theunderside of a projection 15 may be employed.

In use, a cover 10 is placed on a slide 1, as shown in FIGS. 4, 5 and 6to cover the sample 3. The slide 1 will typically be in a tray 21 asshown in FIG. 7, said tray 21 able to hold, for example, 10 slides andcovers of the examples shown. The tray 21 may then be placed into abiological reaction apparatus, such as that disclosed in AustralianProvisional Patent Application No. PS3114/02 by the same applicant,titled “Method and Apparatus for Providing a Reaction Chamber”, filed 20Jun. 2002, and its associated international patent application, filed 20Jun. 2003, the contents of which are hereby incorporated by reference.

Once the tray 21 is loaded into the apparatus (not shown) the slides 1are held in position, typically at an angle of 5 degrees to thehorizontal as shown schematically in FIG. 13 (b), 15 or 17. The cover 10is then moved by an arm (not shown) engaging the locating means 16.Typically, during a sequence referred to as an “open fill”, the cover 10is moved longitudinally along the surface of the slide 1 until thesample 3 is exposed. A fluid is then dispensed by a dispensing means 100such as a probe attached to a pump, onto the fluid receiving zone 13 (asshown in FIG. 13 (b)). The amount of fluid dispensed is typicallysufficient to fill the reaction chamber 24. The use of the cover 10 withthis fill mechanism or methodology allows a small volume of fluid to beuniformly distributed across the reaction chamber 24. Distributing thefluid across the reaction chamber 24 evenly and without bubbles or airspaces allows reactions to take place on the sample 3 with greaterconsistency. Also, dispensing fluid into an empty receiving zone wherethe reaction chamber already contains fluid causes the fluid within thechamber to be replaced by the fluid in the receiving zone minimisingmixing of the fluid in the reaction chamber and newly dispensed fluid.The dimensions of the reaction provide a smooth flow of fluid from thereaction chamber such that there is little mixing of the fluids. This isadvantageous as it allows a previous fluid to be replaced accurately,with minimal original fluid remaining to contaminate later fluids orreactions. This reduces the number of washes required to clear thereaction chamber 24

The volume of fluid in a reaction chamber 24 may be, for example 150microlitres or less, although volumes may vary depending on theapplication and the reaction chamber dimensions.

The reaction chamber 24 is able to retain fluid due to the surfacetension of the fluid, unless additional fluid is added to the fluidreceiving zone, or suction is applied (typically through reduced airpressure) at the end of the slide opposite the fluid receiving zone. Thereaction chamber may be filled as it is formed by the cover 10 beingmoved along the surface of the slide 1 to cover the sample holdingregion 52. Alternatively, the reaction chamber may be filled without thecover being moved relative to the slide, due to the process of capillarywicking of dispensed fluid into the reaction chamber.

In the present examples the cover may be clamped to the slide when notin motion or retracted for an initial fill. The clamping mechanism (notshown) places force around the edge of, for example, cover 10 adjacentthe wall portions 20 to locate the cover 10 with respect to the slide 1during a reaction.

During the withdrawal of the cover 10 from the slide 1 it is sometimesdesirable to remove the cover from contact with the slide. In order toaccomplish this, wings 26 engage the ramps 28 to lift the cover clear ofthe slide. This causes the cover 10 to lift off the slide 1 to preventfluid contact between the slide 1 and cover 10. In this way the slidecan be cleared of virtually all fluid.

Parts of the cover may have different material properties compared tothe properties of the material of the cover body 12, which is typicallyplastic. In one example (not shown) the cavity may have differentmaterial properties, in order to provide a reaction chamber 24 withcertain material properties. A clear plastic material has been found tobe suitable for the body 12 of the cover 10, to provide suitablemechanical properties such as reasonable strength and rigidity. Thecover needs to be sufficiently strong to be moved while clamping forcesare applied to the cover, as the clamping forces assist in providing asealing surface between the walls 20 of the cover 10 and the uppersurface of the slide 1. The cover may be moved to empty or fill thechamber, or also, to promote fluid movement within the reaction chamberto assist a reaction.

The cover should ideally have some flexibility, as it is desirable thatupon application of the clamp, the cavityed face should deflectsomewhat. This has been found to assist in moving the fluid within thereaction chamber and therefore increases the exposure of the sample tothe fluid.

Other properties of the cover 10 include the ability to restrict theheat loss from the surface of the slide 1. Typically the slide will bemounted on a heated block, and the cover will be placed over the sampleon the slide. Heating the slide heats the sample and the fluid in thereaction chamber. If there is excessive heat loss from the cover 10 itis difficult to regulate the temperature of the fluid by beating theslide 1. Further, there may be an excessive temperature gradient acrossthe reaction chamber 24, which is undesirable.

The cavityed face 19, as shown in FIG. 2, may have different surfaceproperties to the rest of the cover. It has been found to be desirableto have similar material properties for the upper surface of the slide 2and the cavity 18. In one example, it is possible to coat the surface ofthe cavity with a material, such as silicon dioxide. This coating may beapproximately 110 nm thick. The coating provides a surface with materialproperties similar to that of a glass slide. It has also been found thatthere are benefits in applying a thin layer (for example 0.5-6 nm) ofChromium Oxide (Cr2O3) to the cavity before applying the silicon dioxidelayer. This application of an intermediate layer between the silicondioxide and plastic provides better adhesion and better thermalexpansion properties for the cavity. Further, coatings in general may beused to improve the flatness of the cavity (which reduce nucleationsites and therefore bubble formation at high temperatures). The coatingsmay be used to modify the capillary flow characteristics of the fluidwithin the reaction chamber, create an impermeable barrier for gas orliquid between the cover and fluid in the reaction chamber, or provide achemically inert surface.

In another example, it is possible to replace the cavityed face 19 witha glass insert supported by the plastic body 12 of the cover 10. It mayalso be possible to change the surface properties of the plastic byplasma discharge.

The covers shown in the examples may be used at temperatures approaching100 degrees Celsius, especially when used for in-situ hybridisationreactions. At higher temperatures, the fluid evaporates and bubbles areproduced. The heating may also cause the cover to bow—the cavity surfaceis hotter than the top of the cover and expands more, causing the cavitysurface to ‘sag’ towards the slide. This helps to remove the bubbles, asthe fluid wants to occupy the smaller spaces more than the bubbles do.The bubbles congregate at the ends of the cavity, and must be allowed toescape.

Experiments have demonstrated that a chamfer at the end of the cavityreliably allows the bubbles to escape to atmosphere. The existingreservoir 17 can be redesigned as illustrated in FIG. 20, where amodified cover 60, similar to that shown in FIG. 14, is shown with achamfer 61 to assist in releasing bubbles, without affecting the evenfluid flow through the cavity 18. The chamfer forms a first angledsection 62 at about 60° relative to the cavity and slide surface.

Fluid evaporation rate is, however, directly linked to the surface areaof the fluid exposed to atmosphere—a larger surface area will evaporatefaster, and require more frequent replenishment. If the bubble escapeangle is steep, the evaporation rate will increase.

This problem can be overcome by using two angles—a shallow angledsection at, say, 15° between the cavity and the chamfer, to minimiseevaporation, leading into the steeper angle for bubble release, whichalso serves to increase the volume of the reservoir.

The cover 60 is also provided with a second, identically shapedreservoir 63 at an opposite end thereof. The second reservoir 63 canalso be used to replenish fluid within the cavity during heating and toallow bubbles to escape. The second reservoir 63 thereby allows forincreased control of fluid conditions within the reaction chamber.

The embodiments of FIGS. 14 and 20 are considered to represent what iscurrently believed to be the best known method of performing the coveraspect of the invention.

1. A cover for a substrate including: a body defining a cavity, forpositioning over the substrate to form a reaction chamber; and aprojection extending from the body to define a fluid reservoir, when thecover is fitted to the substrate, the fluid reservoir being in fluidcommunication with the cavity.
 2. A cover, as claimed in claim 1,wherein the cavity extends the full width of a sample holding region ofthe substrate.
 3. The cover as claimed in one of claim 1 wherein aprotrusion extends from the projection, to assist in wicking fluid intothe reservoir.
 4. A cover as claimed in claim 3, wherein the reservoiris defined by a first section, angled at least at substantially 60°relative to the cavity, and a second section, positioned between thecavity and the first section, and orientated at a reduced angle relativeto the cavity, as compared to the first section.
 5. A cover as claimedin claim 4, wherein the second section is angled at least atsubstantially 15°.
 6. A cover as claimed in any one of claim 1 whereinthe cover is made from a polymer material.
 7. A cover as claimed in anyone of claim 1 wherein the cavity includes a coating of reduced surfaceroughness than the polymer material.
 8. A cover as claimed in claim 7wherein the cavity includes a coating with reduced porosity.
 9. A coveras claimed in claim 7 wherein the cavity has one or more coatings.
 10. Acover as claimed in claim 9 wherein a first coating is a material havingsimilar properties to the material of the slide.
 11. A cover as claimedin claim 10 wherein the first coating is silicon dioxide.
 12. A cover asclaimed in claim 11 wherein a second coating is placed intermediate afirst coating to provide improved contact properties between the coverand first coating.
 13. A cover as claimed in claim 1 wherein the widthof the cavity of the cover is the no larger than the width of amicroscope slide.
 14. A cover as claimed in claim 1, wherein the cavityis substantially planar.
 15. A cover as claimed in claim 1, furtherincluding a locator for controlling and locating the cover, the locatorbeing arranged at an end of the cover opposite the projection.
 16. Acover as claimed in claim 1, further including a second reservoir, at anopposite end of the cover.
 17. A cover as claimed in claim 1, whereinwall portions are located at the edge of the cover, surrounding thecavity on two or more sides.
 18. A cover as claimed in claim 17, whereinthe reservoir is defined between the projection, and legs located oneither side of the cover.
 19. A cover as claimed in claim 18, whereinlegs extend along the sides of the cavity to form the wall portions. 20.A cover according to claim 18 wherein the cover is supported upon thesubstrate on the wall portions.
 21. A covertile according to claim 15wherein the cavity extends to an end edge of the cover adjacent thelocator.
 22. A cover as claimed in claim 1, wherein the cover hasassociated wing structures that allow the cover to be engaged andpivoted relative to the substrate so as to open the reaction chamber andallow the slide to be cleared of fluid.
 23. A combination of a substrateand a cover, as claimed in claim 1, wherein the cavity of the cover isarranged to face the substrate so as to form a reaction chamber.
 24. Amethod of treatment of a sample on a sample holding region of asubstrate including locating a cover, as claimed in claim 1, over thesubstrate, so that the cavity of the cover faces the substrate to form areaction chamber over the sample holding region, and depositing fluidinto the fluid reservoir to allow the fluid to be drawn into thereaction chamber, as required.
 25. A method as claimed in claim 24,further including sliding the cover relative to the substrate to vary adegree of overlap between the cover and the sample holding region, whichresults in a corresponding variation in the reaction chamber volume. 26.A method as claimed in claim 24, further including sliding the coverrelative to the substrate until wing structures associated with thecover are engaged and lifted relative to the substrate to pivot thecover into an open condition, and allow fluid to drain from the reactionchamber.